Recent studies involving cloning and characterization of genes coding for various steroid hormone, thyroid hormone, vitamin D and retinoic acid receptors have indicated that these genes belong to a large family with similar structural and functional properties. Green et al., Nature 320: 134-139 (1986) (human estrogen receptor); Arriza et al., Science 237: 268-275 (1987) (human mineralocorticoid receptor); Hollenberg et al., Nature 318: 635-641 (1985) (human glucocorticoid receptor); Chang et al., Science 240: 324-326 (1988); (human androgen receptor); Lubahn et al., Science 240: 327-330 (1988) (human androgen receptor); Misrahi et al., Biochem. Biophys. Res. Commun. 143: 740-748 (1987) (human progesterone receptor); Weinberger et al., Nature 324: 641-646 (1986) (human thyroid hormone receptor); Baker et al., Proc. Natl Acad. Sci. USA 85: 3294-3298 (1988) (human vitamin D receptor); Giguere et al., Nature 330: 624-629 (1987) (human retinoic acid receptor); Petkovich et al., Nature 330: 444-450 (1987) (human retinoic acid receptor); Robertson, M., Nature 330: 420-421 (1987) (human retinoic acid receptor).
Steroid hormone, thyroid hormone and vitamin D and retinoic acid receptors constitute a class of regulatable transcription factors known as nuclear hormone receptors. Such factors bind high affinity to steroid, hormone or vitamin ligands that enter into target cells. The Hormonal Control of Gene Transcription, Cohen, P. and Foulkes, J. G., editors (Elsevier 1991). Once activated by steroids, hormones or vitamins, these receptors bind to DNA elements, termed "steroid response elements" (SREs), and stimulate initiation of transcription from target promoters.
The amino acid sequence characterizing the nuclear hormone receptor family can be divided into six regions: A-F. Two of these regions, C and E, form domains of known function. C constitutes the DNA-binding domain, and E the ligand-binding domain. Region C has a sequence motif characteristic of DNA-binding zinc fingers.
The DNA-binding domain of asteroid receptor is composed of approximately 80 amino acids that can be divided into two zinc finger units. Rhodes, D. and Klug, A. Scientific American pp56-65 (February 1993). The first zinc finger motif is thought to recognize specific base sequences in the cognate DNA. The second zinc finger motif is believed to function in dimerization of one receptor molecule with another receptor molecule.
Dimerization is required for steroid receptors to interact with DNA. Such receptors bind to DNA as pairs, because each protein in a pair recognizes half of a two-part target site that constitutes a palindrome. That is, each half of a response element is bound by one molecule in a receptor dimer. To bind successfully, the receptor must be able to distinguish both the specific base sequence of the half site and the spacing, i.e., the number of base pairs per half site.
The parts of the three-dimensional structure of the DNA-binding domain responsible for recognizing DNA and pairing with another receptor molecule have been elucidated. Each of the two zinc finger units consists of two parts: an irregular loop followed by an alpha helix. Each irregular loop is formed by the combining of four amino acids (e.g., cysteines) with a single zinc ion. The two fingers merge into a single arrangement where the two helices form a cross due to the mutual attraction of hydrophobic amino acids.
The amino acids that are critical for recognizing specific bases fall on the helix in the first unit. One alpha helix from each DNA binding domain thus makes contact with the bases of a DNA double helix. The function of the second helix is probably to serve as a backing strut to hold the recognition helix in place. Those amino acids that are responsible for forming a dimer reside in the irregular loop of the second unit. The pairing of the receptors through the dimerization region is thought to orient the dimer. That is, the two recognition helices on the dimer are arranged by dimerization so that the spacing between them matches the spacing defining the half site in the appropriate SRE.
Genomic cloning of human estrogen receptor sequences indicates that this gene is about 140 kb in length and is divided into eight exons. The N-terminal A/B region of the receptor is almost entirely encoded within the first exon. The DNA binding domain, region C, is encoded by exons 2 and 3. Exon 2 codes for the first zinc finger of the DNA binding domain, and exon 3 the second zinc finger. The "hinge" region, which is region D, is assembled by exon 4. The hormone binding domain, region E, is encoded in five exons, exons 4-8. The end of exon 8 encodes region F. These conclusions are based on the genomic organization of estrogen receptor genes from human (Ponglikitmongkol et al., EMBO J 7: 3385-3388 (1988)) but can be extended to other species and steroid hormone receptors.
The hallmark of steroid hormone receptors is their capacity to bind steroid hormonal ligands. There are five families of steroid hormones that can be classified on a structural and functional basis. Norman, A. W. and Litwack G., Hormones (Academic Press 1987). They are the estrogens (such as estradiol), the androgens (such as testosterone), the progestins (such as progesterone), the mineralocorticoids (such as aldosterone), and the glucocorticoids (such as cortisol). All of these steroid hormones are biologically derived from cholesterol.
The sex hormones of vertebrates, steroids derived from cholesterol in the gonads and adrenal cortex of both sexes, include the estrogens, the androgens, and the progestins. The estrogens and androgens are important in various aspects of growth and morphological differentiation, as well as in the development and regulation of sexual and reproductive behavior and cycles. Goodman, H. M., Basic Medical Endocrinology (Raven Press 1988). Estrogens predominate in the female, and androgens in the male. The production and secretion of these steroids is under the control of the gonadotropins, follicle stimulating hormone (FSH) and luteinizing hormone (LH), which are synthesized and secreted in the pituitary. These gonadotropins are, in turn, regulated by gonadotropin releasing hormone (GnRH), which is produced and released in the hypothalamus.
The estrogens are a family of steroids produced in the vertebrate ovary, testis, and adrenal cortex. Cholesterol is converted to progesterone, which is then transformed to the androgens, androstenedione and testosterone. The estrogens, of which estradiol-17.beta. is the most potent, are made from these androgens.
The importance of estrogen in development and sexual differentiation is unresolved. Prenatal sexual differentiation has been shown to be dependent upon the secretion or absence of androgens during a critical period in development. Without androgens, the female configuration develops with the Mullerian ducts retained and the Wolffian ducts eliminated. In the presence of androgens, the male tract develops. By comparison, a role for estrogen during embryogenesis has been merely postulated. Estrogen synthesis is reported to be activated in male and female embryos at the time of blastocyst implantation in the uterus (George, F. W. and Wilson, J. D., Science 199: 200-202 (1978); Dickman et al., Science 195: 687-688 (1977)). A recent study, using a very sensitive reverse transcriptase-polymerase chain reaction technique, has detected estrogen receptor mRNA in blastocyst and 2-cell stage embryos (Hou, Q. and Gorski, J., Proc. Nat. Acad. Sci. USA (in press-MS#V1316)). Although estrogen is known to act via the estrogen receptor to mediate the sexual differentiation of the brain, its mode of action is not understood. (Gorski, R., Endocrinology 133: 431 (1993)).
A chemical interplay among estrogen, the gonadotropins, and GnRH occurs that is most apparent in the mammalian female and that begins, in humans, at menarche and continues until menopause. Norman, A. W. and Litwack G., Hormones (Academic Press 1987). It is known as the female reproductive cycle. Cyclical changes in FSH, LH, progesterone, estradiol, and GnRH are accompanied by morphological changes of the uterine endometrium. The cycle can be conveniently divided into two phases: pre- and postovulatory. The key events of the preovulatory phase are growth and maturation of the ovarian follicle and maturation of the uterine endometrium. The key event of the postovulatory phase is the growth, development, and involution of the corpus luteum and, in the absence of initiation of pregnancy, the shedding of the uterine endometrium. A 28-day cycle is generally regarded as the mean length of a normal menstrual cycle.
Throughout the menstrual cycle there is a changing ratio of FSH to LH. At the beginning of the follicular phase, from days 1 to 10, FSH blood levels exceed LH levels. At approximately day 10 there is a crossover, and LH rapidly becomes elevated to a very large peak or surge on the day of ovulation, day 14. Blood levels of FSH are increased at ovulation, but not to the extent achieved by LH. Throughout the luteal phase the levels of FSH are low, while LH levels are relatively high on days 14-18. LH levels fall to low levels in the absence of fertilization by day 28.
The secretions of FSH and LH are both believed to be governed by the same hypothalamic releasing factor, GnRH. This makes it difficult to devise biochemical mechanisms that can explain the changing ratio of FSH to LH throughout the menstrual cycle. One suggestion is that the response of the adenohypophysis cell which secretes FSH and LH in response to the hypothalamic-derived GnRH is determined by blood concentrations of estradiol and progesterone. There is evidence for the existence of receptors for both estrogen and progesterone in the adenohypophysis, the hypothalamus, as well as in higher brain centers. Changing occupancy of all these receptors by estrogen and progesterone could have the consequence of modulating the ratio of FSH to LH which is secreted.
The dominant hormonal changes of the menstrual cycle, particularly on the uterine endometrium, are mediated by the steroid hormones secreted by the ovaries and corpus luteum. Associated with the cyclical changes in the gonadotropins described above are related changes in the blood levels of estrogen and progesterone. In the early preovulatory phase, estradiol levels remain low until .sup..about. 7-8 days before the LH surge characterizing ovulation (day 14). Then estradiol increases and reaches a peak 1 day before the LH surge. During the postovulatory phase, there is a drop of estradiol at days 14-16 followed by a rise to a second peak at day 22-23.
Progesterone secretion by the ovaries in the follicular period is very low and accordingly the blood levels are low. Some additional progesterone is produced by the adrenals. The blood levels of progesterone rise dramatically in the luteal period after ovulation and peak at days 18-24 of the cycle. This rise and peak coincides with maximal steroid hormone synthesis of the corpus luteum. The blood levels of both progesterone and estradiol fall after day 24 until initiation of menstruation. If fertilization occurs, the corpus luteum is rescued and continues the production of progesterone until the placenta becomes functional.
In the ovary, the follicle matures and the corpus luteum develops. In a select follicle, at the beginning of the cycle, the oocyte becomes progressively larger and the surrounding granulosa cells proliferate. The granulosa cells produce principally only estradiol. The cells adjacent to the follicle become enlarged and arranged in concentric circles in a cellular array termed the theca. The theca cells are active in steroid metabolism and can produce both estradiol and androstenedione. As the follicle develops, the granulosa cells produce, due to the trophic actions of FSH, increasing amounts of estradiol. Blood levels of estradiol increase slowly and then more rapidly to reach an apex just prior to the LH surge. This increase has the effect of establishing the high levels of estradiol required for positive feedback at the hypothalamus and pituitary level. Simultaneously, receptors for LH are appearing in increasing concentration on both the thecal and granulosa cells in preparation for ovulation. Very late in the follicular phase, after blood levels of LH are greater than blood levels of FSH, the thecal cells are stimulated by LH to begin producing progesterone. The general maturation of the follicle is complete and it is referred to as a graafian follicle.
After ovulation, both the thecal and granulosa cells of the follicle undergo rapid mitosis. Out of the follicle a new endocrine organ is created, the corpus luteum. The corpus luteum reaches its maximum size by the middle of the luteal phase, and if fertilization does not occur it undergoes regression and degeneration. The corpus luteum under the stimulus of LH actively produces progesterone.
The uterine endometrial epithelium undergoes morphological changes throughout the 28 days of the menstrual cycle. During the first half of each menstrual cycle, when estrogen concentrations gradually increase to reach a maximum 24 hr before ovulation and while progesterone concentrations are relatively low, the endometrium is stimulated to increase in thickness. Then 36 hr after ovulation, when the progesterone concentration rises sharply due to its secretion from the corpus luteum and estrogen levels are maintained at levels two-thirds of their previous maximum, further specific morphological changes are induced in the endometrium. There is stromal edema, glandular secretion, and vascularization. In the event that fertilization does not occur, the stromal edema decreases, glandular secretions diminish, and vascularization ceases. At the time of the rapid fall in estrogen and progesterone at the end of the luteal phase, blood stasis and stromal degeneration occur and the endometrial tissue is sloughed off.
The steroid contraceptive pill is the most widely used method of contraception; worldwide some 50 million women take some form of oral contraceptive. The most common of the presently employed synthetic oral contraceptives is composed of tablets containing both a progestin and an estrogen. Contraceptive steroids prevent ovulation by interfering with the release of GnRH by the hypothalamus and LH and FSH by the pituitary and thus block the midcycle surge of gonadotropins which mediate ovulation.
Estrogen is seen to play a central role in normal female physiology. The dominant actions of estrogen occur in the female reproductive tract, although there are also significant biological actions mediated in the pituitary, hypothalamus and brain as well as in a variety of other visceral organs. Estrogen receptors are distributed among a number of tissues, including uterus, vagina, placenta, oviduct, ovary, corpus luteum, mammary tissue, pituitary, hypothalamus, brain, liver, kidney, lung, bone, etc. The presence of estrogen receptors in a tissue presumes biological actions of estrogen at that location. Indeed, an effect by estrogen in bone has recently been explained (see infra).
In female pathology, a connection between estrogen and cancer, for example, breast and uterine cancer, is well documented but poorly understood. An association between estrogen receptor action and infertility has been suggested. Estrogen is important in protection against osteoporosis and cardiovascular disease, albeit via unknown mechanisms.
Hormone replacement therapy for the reversal of changes associated with decreased levels of estrogen as characterizes menopause has certain risks and benefits. Gambrell, R. D., American Family Physician 46: 87S-96S (1992); Harlap, S., Am. J. Obstet. Gynecol. 166: 1986-1992 (1992); Whitcroft and Stevenson, Clinical Endocrinology 36: 15-20 (1992). Estrogens are effective in postmenopausal women in reversing vasomotor symptoms and vaginal atrophy. Estrogens also prevent the bone loss associated with osteoporosis. Further, estrogens are known to reduce the risk of cardiovascular disease. Therapy unopposed by progesterone treatment, however, increases the risk of developing endometrial cancer, breast cancer, and, possibly, certain other cancers.
Post-menopausal osteoporosis affects 1.5 million women each year, making this disease a major health care problem. Horowitz, M. C. Science 260: 626-627 (1993). The pathological bone loss underlying this disorder can be prevented by estrogen replacement therapy. The mechanism by which estrogen exerts its bone sparing effect is unclear. Recent advances, however, indicate that estrogen regulates the circuitry of cytokine action that controls bone remodeling.
In normal bone remodeling, bone resorption by osteoclasts is balanced against bone formation by osteoblasts. Osteoclast mediated bone resorption can be influenced by two processes: activation, in which the resorptive function of mature osteoclasts is increased, and recruitment, in which osteoclast progenitors are stimulated to become mature cells. Activation occurs when certain cytokines, such as parathyroid hormone (PTH) or peripheral blood monocyte (PBM)-derived interleukin-1 (IL-1) or tumor necrosis factor (TNF), stimulate osteoblasts to secrete other cytokines that act on osteoclasts to cause bone resorption. In recruitment, still other cytokines are required for the maturation of progenitors into osteoclasts. These cytokines, such as macrophage-colony stimulating factor (M-CSF) and interleukin-6 (IL-6), are secreted by stimulated osteoblasts, while other cytokines, such as granulocyte macrophage-colony stimulating factor (GM-CSF), are secreted by PBMs.
It is believed that with estrogen acting as the inhibitor, PBMs serve as a source of limited quantities of IL-1, TNF, and GM-CSF, and osteoblasts secrete small amounts of M-CSF and IL-6. In this way, normal bone remodeling is maintained. The absence of estrogen is thought to cause PBMs to increase secretion of IL-1, TNF, and GM-CSF. Osteoclast activation and differentiation are thereby enhanced. Normal bones can consequently become osteoporotic.
In the estrogen deficient situation, a direct role for osteoblasts is also postulated. Osteoblasts probably secrete more IL-6, causing increased osteoclast differentiation, and probably also produce-more activating cytokines that directly activate osteoclasts. Bone resorption is presumably augmented, and osteoporosis ensues. The finding that estrogen receptors are expressed by macrophages and bone cells (Eriksen et al., Science 241: 84-86 (1988) (osteoblasts); Pensler et al., J. Bone Mineral Res. 5: 797-802 (1990) (osteoclasts)) corroborates the theory that estrogens exert osteoporotic effects by regulating bone cell cytokines.
The classical strategy for the identification of endocrine glands and hormones has been ablation of the suspected tissue, which should produce deficiency symptoms, followed by replacement of the ablated tissue, which presumably reverses the deficiency symptoms. The study of steroid hormones, including sex hormones, such as the estrogens, has profited by the application of such methods. Nevertheless, a modern approach, tailored to address the regulatory capacity of the steroid receptor in physiological responses, is needed to supersede the classical.
In particular, a need exists for an animal model system to study the biological role of hormones, including steroid hormones and sex steroids, like the estrogens, in growth, development, morphological differentiation, sexual and reproductive behavior and cycles, etc. Such an animal model system should provide a means for testing sex hormones and synthetics that mimic or antagonize sex hormones for use in birth control methods. It should also facilitate evaluation of hormone replacement therapies that raise questions about risks and benefits. The animal model system should be amenable to characterization of materials suspected of precipitating or conferring protection against, e.g., osteoporosis, cardiovascular disease, breast cancer, endometrial cancer, and other cancers, and thus contribute to diagnosis, prognosis, and therapy of these major diseases. Accordingly, it is an object herein to provide certain mutant non-human vertebrates having a deficit of functional steroid hormone receptors, and related methods and constructs.